Coated metal surface on solid support for analyte detection by displacement

ABSTRACT

A coated metal surface on a solid support, wherein the coating consists of a protein layer firmly attached to the metal surface, and said protein layer is coupled to linker molecules that are bound to low molecular weight antigens, wherein the linker molecules are coupled to the protein layer and are bound to the antigen via functional end groups and contain between the functional end groups an aliphatic hydrocarbon of 1, 2 or 3 carbon atoms, and wherein the antigens are optionally reversibly bound to antibodies specific for the antigens, is described. The coated metal surface on a solid support may be used in a method of detecting analyte antigens as part of an analysis device, such as a Piezoelectric Crystal Microbalance device or a Surface Plasmon Resonance biosensor, for detection in an aqueous solution of an analyte antigen with higher affinity to an antibody than the antigen of the coating by monitoring the displacement of the antibody from the coating.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. 119(e) of U.S.Provisional Patent Application Ser. No. 60/389,496 filed Jun. 19, 2002,the whole of which is incorporated herein by reference.

The present invention relates to a coated metal surface on a solidsupport useful in analyte detection in an aqueous solution bydisplacement of reversibly bound antibodies specific for the analytefrom the metal surface coating. Detection of the displacement, and thusthe presence of the analyte in an aqueous solution, is performed with ananalysis device, such as a Piezoelectric Crystal Microbalance (PCM)device or a Surface Plasmon Resonance (SPR) biosensor.

BACKGROUND

The SPR biosensor is a sensitive real-time technique, which can be usedto extract information about molecular interaction near certain metalsurfaces. It offers the possibility to determine concentration,association and dissociation rate constants and affinity as well asepitope mapping and determination of interaction specificity [B.Liedberg and K. Johansen, Affinity biosensing based on surface plasmondetection in “Methods in Biotechnology, Vol. 7: Affinity Biosensors:Techniques and Protocols”, K. R. Rogers and A. Muchandani (Eds.), HumanaPress Inc., Totowa, N.J., pp. 31-53]. One of the componentsparticipating in the studied reaction is immobilized on the metalsurface either before or during the SPR experiment. The immobilizedmolecule is exposed to a continuous flow into which one can injectinteracting species. The method is based on optical detection and thesensing signal reflects changes in dielectric function or refractiveindex at the surface. These changes can be caused by molecularinteraction at the surface.

The PCM technique is based on an oscillating piezoelectric crystal in amicrobalance device, wherein the crystal consists of e.g. quartz,aluminum nitride (AlN) or sodium potassium niobiates (NKN. When thecrystal is a quarts crystal, the device is referred to as a QCM (quartzcrystal microbalance). The PCM and QCM are gravimetrical sensors and arethus sensitive to mass changes. A QCM comprises a piezoelectric quartzcrystal plate upon which metal electrodes have been deposited on bothsides. An alternating potential difference applied on such a crystalplate induces shear waves. At certain frequencies—such that thethickness is an odd integer of half wavelengths—the crystal will be inresonance [M. Rodahl, F. Höök, A. Krozer, P. Brzezinski and B. Kasemo,Quartz crystal microbalance setup for frequency and Q-factormeasurements in gaseous and liquid environments, Review of ScientificInstruments 66 (1995) pp. 3924-3930] and [Saurbrey,Z.Phys. (1959),pp155, 206-222]. The energetically most favourable number of halfwavelengths is one. The resonance frequency is dependent on thethickness of the crystal, but is normally in the MHz range. A masschange on the surface of the plate will result in a shift in theresonance frequency. The fact that frequency shifts of 0.01 Hz can beeasily measured makes the QCM a sensitive sensor for determining massvariations. A number of patents and other publications describe the useof piezoelectric quartz crystals (QCM) as affinity-based chemicalsensors/detectors in e.g. various immuno-assay techniques, and detectionof bacteria and virus. In most of these applications the QCM-instrumentis used to analyze the weight gain of the crystal after interactionbetween antibodies and antigens.

The crystal is used as a microbalance to measure very small masses. Thethin piezoelectric sensor crystal electrode used in our experiments hasgold evaporated on each side. The crystals can be made to oscillate atits resonance frequency by applying an AC-voltage over the electrode.The principle behind the QCM-technique is that the resonant frequencychanges when the mass of the crystal changes. By using this method themass changes in a bio-molecule layer of a crystal can be monitored. Manystudies have been reported utilizing the QCM, where the crystal has beencoated with a coating that interacts in a specific way with a moleculeor particle, e.g. a bacterium, virus, antibody or antigen, resulting ina loss or gain of weight of the crystal, which change in weight ismeasured.

There are obvious difficulties in analyzing small molecules withconventional immunosensors due to the low response, i.e. small change inweight of the sensor crystal. For attaining the necessary detection ofsmall molecules, the sensitivity of the system has to be improved. Thismay be achieved by using displacement reactions where a large antibodymolecule is detached from the sensor surface by dissociation andreaction with an analyte antigen that has a higher affinity to theantibody than the antigen bound to the sensor surface.(Willner et. al EP0 843 816).

DESCRIPTION OF THE INVENTION

The present invention provides a coated metal surface on a solid supportthat is useful in an analysis device for detection of an analyte antigenin an aqueous solution by monitoring displacement of a reversibly boundantibody from the coating by dissociation and reaction with the analyteantigen.

In this specification and claims the word antibody is intended tocomprise whole antibodies or antigen-binding parts of antibodies orsynthetic antigen-binding molecules.

Thus, one aspect of the invention is directed to a coated metal surfaceon a solid support, wherein the coating consists of a protein layerfirmly attached to the metal surface, and said protein layer is coupledto linker molecules that are bound to low molecular weight antigens,wherein the linker molecules are coupled to the protein layer and arebound to the antigen via functional end groups and contain between thefunctional end groups an aliphatic hydrocarbon of 1, 2 or 3 carbonatoms, and wherein the antigens are optionally reversibly bound toantibodies specific for the antigens.

The firm attachment of the protein layer to the metal surface may beaccomplished by contacting the protein, e.g. albumin, casein, variousglobulins, LDH, ovalbumin, with the cleaned metal surface whereby theprotein adheres to the surface, or the protein may be immobilized onto apre-deposited functional surface (containing e.g. tiol, carboxylic acidand/or amino groups), or by grafting onto the surface by cross-linkingto a polymer structure on the surface.

The linker molecules are coupled to the protein layer by reactionbetween functional end groups on the linker molecules, such as tiol,carboxylic acid, amino, and hydroxide groups, and functional groups onthe protein, such as tiol, carboxylic acid, hydroxyl groups and aminogroups.

The linker molecules are usually at first bound to the low molecularweight antigens by reaction between functional end groups on the linkermolecules, such as tiol, carboxylic acid, amino and hydroxyl groups orleaving groups, e.g. halides, mesylates, tosylates, activated carboxylicacids, e.g. acid anhydrides and acid chloride, and functional groups onthe antigens, such as amino, keto, and hydroxyl groups. It may benecessary to introduce or create a reactive functional group on the lowmolecular weight antigen prior to the reaction, e.g. in case the antigenlacks functional groups for the reaction.

An important feature of the linker molecule in the coating of thepresent invention is that it has, in addition to the functional endgroups for reaction between the protein layer and the antigen, a shortaliphatic hydrocarbon chain of 1, 2 or 3 carbon atoms. If the linkergroup in the coating is longer than 3 carbons, the affinity to theantibody is too high so that only a limited displacement of the antibodycan be monitored upon exposure to the analyte antigen in aqueoussolution, which decreases the sensitivity.

The coated metal surface on a solid support according to the inventionwill usually be stored separately from the antigen-specific antibodiesprior to use. When used in displacement analysis, the coated metalsurface on a solid support will, however, comprise the specificantibodies reversibly bound to the antigens of the coating.

The metal of the coated metal surface on a solid support according tothe invention is preferably selected from the group consisting of gold,silver, aluminum, chromium and titanium. The presently preferred metalis gold.

The antigen of the coating is the same as or a derivative of the analyteantigen except that it is immobilized through a bond to the linkermolecule. The antigen of the coating may thus be derivatized to modulatedissociation of the bound antibody in an aqueous solution.

The antigens bound to the linker molecules of the coating according tothe invention are the same or different and are bound to the sameprotein layer or to different patches of protein layers, i.e. theantigens may bind to the same specific antibodies or there may be amixture of two or more bound antigens binding to different specificantibodies enabling the detection of the presence of several differentanalyte antigens in an aqueous solution. In case the antibodies carrydifferent markers, such as fluorescent markers, it will be possible todetect displacement of the different antibodies. However, a mixture ofseveral different antibodies will normally be used in cases wherescreening of samples for any of the target antigens is sufficient, suchas screening of samples for any narcotics or explosives. The differentantibodies may be kept apart from each other by coating the metalsurface with discrete patches or microarrays of spots of proteinscarrying different antigens. In a preferred embodiment of the inventionthe antigen of the coating is selected from the group consisting ofoptionally derivatized explosives and narcotics. In case the selectedantigen of the coating binds too strongly to the specific antibody sothat the dissociation of the antibody in aqueous solution is hampered,the antigen molecule may be chemically modified, e.g. by modification offunctional groups such as ester or amino groups (by removal of, orreplacing the original groups) or by eliminating a part of the antigenmolecule, or introducing new functional groups or side chains to theantigen molecule, to reduce its affinity to the antibody.

The explosives are preferably selected from the group consisting oftrinitrotoluene (TNT), dinitrotoluene (DNT),hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX),octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazine (HMX), pentaerythritoltetranitrate (PETN), and nitroglycerine (NG), and the narcotics arepreferably selected from the group consisting of cocaine, heroine,amphetamine, methamphetamine, cannabinols, tetrahydrocannabinols (THC),and methylenedioxy-N-methylamphetamine (Ecstacy).

In a presently preferred embodiment the solid support of the coatedmetal surface on a solid support according to of the invention is apiezoelectric crystal electrode or a glass plate or prism. In case thecrystal is a quartz crystal, the coated metal surface on thepiezoelectric quarts crystal electrode is suitable for use in a QCMdevice, whereas the coated metal surface on a glass plate or prism issuitable for use in a SPR apparatus.

Another aspect of the invention is directed to the use of the coatedmetal surface on a solid support according to the invention as part ofan analysis device for detection in an aqueous solution of analyteantigens with higher affinity to specific antibodies than the antigensof the coating by monitoring the displacement of the antibodies from thecoating.

Yet another aspect of the invention is directed to a method of detectinganalyte antigens in an aqueous solution comprising activating, ifnecessary, the coated metal surface on a solid support according to theinvention lacking bound antibodies by bringing antigen-specificantibodies into contact with the coated metal surface in an aqueoussolution, allowing binding of the antibodies to the antigens of thecoating, removing excess antibodies, bringing the aqueous solutionpossibly containing the analyte antigens that have higher affinity tothe antibodies than the antigens of the coating into contact with theantibodies reversibly bound to the coating, allowing the antibodies todissociate and react with the analyte antigens, and detecting the lossof mass on the coated metal surface by means of an analysis device. Inan embodiment of the method of the invention the analysis device isselected from the group consisting of a Piezoelectric CrystalMicrobalance device and a Surface Plasmon Resonance biosensor. Thepiezoelectric crystal is e.g. of quarts, aluminum nitride (AlN) orsodium potassium niobiates (NKN).

In a presently preferred embodiment the analysis device comprises a flowcell in which the coated metal surface on a solid support according tothe invention is placed.

The invention will now be illustrated by some drawings and descriptionof experiments, but it should be understood that the invention is notintended to be limited to the specifically described details.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chemical formula of some narcotics, Heroine,Amphetamine, Ecstacy, and Methamphetamine, and narcotics-linkermolecules, Heroine-linker molecule, Amphetemine-linker molecule,Ecstacy-linker molecule and Morphine-linker molecule.

FIG. 2 shows the chemical formula of some additional narcotics,Cannabinol, Tetrahydrocannabinol and Cocaine, and a Cocaine-linkermolecule.

FIG. 3 shows the chemical formula of some explosives,2,4,6-Trinitrotoluene (TNT) and 2,4-Dinitrotoluene (2,4-DNT).

FIG. 4 is a schematic figure showing a typical coating of anantigen-linker molecule conjugated to a protein that is attached to agold surface. The binding of the antigen via the linker molecule to theprotein is highlighted.

FIG. 5 shows the relative frequency change upon antibody injectionagainst cocaine (0.02 mg/ml) and subsequently injection of cocaine (10pg/μl). The flow rate is 50 μl/min and the injection volume (loopvolume) 100 μl.

FIG. 6 shows the relative frequency change upon antibody injection andsubsequently injection of TNT (10 pg/μl and 100 pg/μl). The flow rate is250 μl/min.

GENERAL DESCRIPTION OF EXPERIMENTS

The experiments were conducted in a QCM-system having electrodesoptimized to improve the sensitivity of the analysis of low molecularweight compounds. The immunosensing system is based on a principle ofdisplacement.

The immunoassay system that consists of an electrode, which isfunctionalzed with an antigen derivative immobilized to a gold surfaceof the electrode via a short linker molecule and a protein layer.Monoclonal antibodies against the antigen was then introduced to theantigen functionalized electrod.

The monoclonal antibodies against the antigen were prepared byimmunization of mice with the same antigen linked, by a longer link thanthe 1-3 carbon atom linker used for the coating of the presentinvention, to KLH (Keyhole Limpet Hemocyanin). The antibody synthesesprocedure is a well-known procedure. [see e.g. Hybridoma Technology inthe Biosciences&Medicine. T. A. Springer, editor, Plenum Press, 1985.]The antibodies used in our analysis exhibit sub-nanomolar affinity tothe antigen with very defined specificity.

When the electrode is exposed to the free antigen (analyte) in solutiona decrease in weight due to a displacement of the surface boundantibodies can be seen. This decrease in weight of the electrode ismonitored by an increase in the oscillation frequency of the crystal(FIGS. 5,6).

In the present experiments the antibodies against the antigens to bedetected (analyte antigens) are first physically bound to the surfacelocalized antigen derivatives, i.e. the antigens of the coating of theinvention, by weak interactions. When these weakly boundantibody/antigen complexes are exposed to the antigen in solution, theanalyte antigen, a fraction of the weakly bound antibodies will leavethe surface coating, due to a stronger affinity to the analyte antigenin solution. This will reduce the mass of the QCM-crystal giving anincrease of the frequency. The concept is based on the fact that theantibody has a higher affinity to the antigen (analyte) in solutioncompared to its affinity to the surface localized antigen (highdissociation constant, Kd). It is of utmost importance that the affinitybetween the antibody and the surface is optimized to give a weight lossupon a contact with a low concentration of the analyte antigen withouttoo much weight loss upon contact with the buffer containing no antigen.It is therefore very important to optimize the affinity of theantibodies to the surface bound antigen.

The trade off in the method is the level of background signal. When asurface bound antibody has too high affinity to the immobilized antigenthe displacement of the antibody by free antigen is very difficult and alarger amount of free antigen is needed to displace the antibody fromthe surface. On the other hand, if the surface bound antibody exhibitstoo low affinity towards the surface bound antigen the antibody willleave the surface too fast even in the absence of free antigen. Thedynamic in the weak biological interaction in such a system isintriguing, binding and release are occurring simultaneously and it isnot that easy to predict and optimize a system for technical use in abiosensor.

The quartz crystal electrodes are coated with a metal e.g. gold, whichis chemically modified in order to minimize the adsorption of proteins,especially antibodies. In addition, the surface layer is alsofunctionalized with an antigen-derivative which functions as a weakantigen for the antibody of interest. It is very important that noadsorption of other antibodies is occurring (non-specific adsorptions).A number of various surface modifications have been tested to find anantigen-functionalized surface coating, which is protein-repellent (nonon-specific adsorption).

We have tested the method by grafting antigen structures relating totrinitrotoluene (TNT), dinitrotoluene (DNT), heroine, amphetamine,methamphetamine, methylenedioxy-N-methylamphetamine (extacy),tetrahydrocannabinol (THC) and cocaine onto various proteins, that wereused to modify the electrode surface in the QCM-apparatus. The antigenswere grafted on the amino groups on the proteins via a carboxylic acidgroup on a linker bound to the antigen.

DETAILED DESCRIPTION OF EXPERIMENTS

Conjugation of Cocaine to a Protein e.g. Various Albumins GammaGlobulins, and Other Well Characterized Proteins.

In general we conjugated the antigens to the proteins by using the watersoluble carbodiimide EDC [(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide] along with N-hydroxysuccinimide (NHS) to create activeester intermediates of the carboxylated antigen derivatives (e.g. thecarboxylated drug-linker molecules in FIGS. 1 and 2). The activatedNHS-esters of the antigens were then added to an aqueous solution of theprotein, subsequently dialyzed against buffer.

The antigen (20 μmole) was reacted with a 60 μmole NHS and 50 μmole EDCin dimethylformamide (0.5 ml) for 2 h at room temperature. The protein(20 mg) (e.g. albumin) was dissolved in 3 ml 0.1 M NaHCO₃, pH 8. TheDMF-solution of the activated cocaine was mixed with the proteinsolution during 4 h at room temperature, subsequently dialyzed againstbuffer in 24 h.

Coating of QCM-electrodes

The gold surface of the piezoelectric electrode is washed with varioussolvents and water prior to soaking the electrode in a solution of theprotein conjugate (conc. 0.1-3 mg/ml) for 1 h at room temperature,subsequently washed thoroughly in distilled water and allowed to dry.

Measurement Procedure

The coated piezoelectric electrode was fixed in a flow through cell withan internal volume of approximately 10 μl. The cell volume is sealedwith a soft rubber O-ring and only one side of the electrode is incontact with the flowing system. The flow rate is adjusted between 5-500μl/minute (phosphate buffer, PBS). The samples are injected via a loop(50-100 μl) to the cell (flow rate of between 5-500 μl/minute). Thereaction (weight gain or weight loss) of the surface modified electrodeis continuously monitored as a measurement of the change in frequency.

Results

When injecting an antibody (concentration 0.02 mg/ml, loop volume 0.1ml) against cocaine a selective adsorption of the antibody immediatelyoccurred resulting in a pronounced decrease in the frequency (see FIG.5). When a buffer solution continuously is flowing through the cellafter the antibody injection, a slight increase of the frequency oftenis observed, which indicates a slight loss of antibody. However, wheninjecting a sample of cocaine (concentration of 10 pg/μl and 100 pg/μl,loop volume 0.1 ml) an increase in frequency can be observed.

The same pattern is seen in FIG. 6, when injecting antibody against TNTand injecting TNT-samples.

A series of conjugates between cocaine derivatives, i.e. cocaine boundto linker molecules, and a protein, albumin, were made. One of thecocaine-linker molecules is shown in FIG. 2. Only the derivatives havingshorter aliphatic chains than 4 carbon atoms, in addition to possiblecarbon atoms in the functional end groups (carboxylic acid group in FIG.2), show a significant displacement of antibody on exposure to theanalyte (i.e. cocaine).

Also other cocaine derivatives having long linkers bound to the moleculewere tested and resulted in no displacement of the antibody at exposureto the free cocaine in solution. In conclusion, from these experimentsit is evident that the affinity between the immobilized cocaine antigenand its antibodies was too high to be useful in the displacementanalysis when using long linkers.

Heroine antigens were functionalized in similar way on its OH-groups andN-group. The results clearly showed a pronounced increase in detecteddisplacement when the linker molecule had less than 4 carbon atoms.

Observations made with Final Antigen/Antibody Coated Metal Surfaces ofthe Invention:

-   -   1. Very little non-selective adsorption of antibody compared to        the selective antibody.    -   2. Active in aqueous solution for the displacement reaction for        a long time (e.g. >8 h,RT, in a flow cell)    -   3. Minor desorption (base line drift) of antibody when no        analyte is present. (e.g. 0.5%/min in aqueous solution in a flow        cell)    -   4. Stable in dry state when antigen loaded (>3 months at room        remperature (RT).

The invention claimed is:
 1. A coated metal surface on a solid support,wherein the coating consists of a protein layer firmly attached to themetal surface, and said protein layer is coupled to linker moleculesthat are bound to low molecular weight antigens, wherein the linkermolecules are coupled to the protein layer and are bound to the antigenvia functional end groups and contain between the functional end groupsan aliphatic hydrocarbon chain of 1, 2 or 3 carbon atoms, and whereinthe antigens are reversibly bound to antibodies specific for theantigens.
 2. The coated metal surface on a solid support according toclaim 1, wherein the metal is selected from the group consisting ofgold, silver, aluminum, nickel, chromium and titanium.
 3. The coatedmetal surface on a solid support according to claim 1, wherein theantigens are the same or different and are bound to the same proteinlayer or to different patches of protein layers and are selected fromthe group consisting of optionally derivatized explosives and narcotics.4. The coated metal surface on a solid support according to claim 3,wherein the explosives are selected from the group consisting oftrinitrotoluene (TNT), dinitrotoluene (DNT),hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazine (HMX), pentaerythritol tetranitrate (PETN),and nitroglycerine (NG).
 5. The coated metal surface on a solid supportaccording to claim 3, wherein the narcotics are selected from the groupconsisting of cocaine, heroineheroin, amphetamine, methamphetamine,cannabiols, tetrahydrocannabiols (THC), andmethylenedioxy-N-methylamphetamine (Ecstacy).
 6. The coated metalsurface on a solid support according to claim 1, wherein the solidsupport is a piezoelectric crystal electrode or a glass plate or prism.7. A method of detecting analyte antigens in an aqueous solutioncomprising activating the coated metal surface on a solid supportaccording to claim 1 lacking bound antibodies by bringingantigen-specific antibodies into contact with the coated metal surfacein an aqueous solution, allowing binding of the antibodies to theantigens of the coating, removing excess antibodies, bringing theaqueous solution possibly containing the analyte antigens that havehigher affinity to the antibodies than the antigens of the coating intocontact with the antibodies reversibly bound to the coating, allowingthe antibodies to dissociate and react with the analyte antigens, anddetecting the loss of mass on the coated metal surface by means of ananalysis device.
 8. A method according to claim 7, wherein the analysisdevice is selected from the group consisting of a Piezoelectric QuartzCrystal Microbalance device and a Surface Plasmon Resonance biosensor.9. The method according to claim 7, wherein the analysis devicecomprises a flow cell in which the coated metal surface on a solidsupport is placed.
 10. The method according to claim 8, wherein theanalysis device comprises a flow cell in which the coated metal surfaceon a solid support is placed.
 11. A coated metal surface on a solidsupport, wherein the coating consists of a protein layer firmly attachedto the metal surface, wherein the metal is selected from the groupconsisting of gold, silver, aluminum, nickel, chrome chromium andtitanium, and said protein layer is coupled to linker molecules that arebound to low molecular weight antigens, wherein the linker molecules arecoupled to the protein layer and are bound to the antigen via functionalend groups and contain between the functional end groups an aliphatichydrocarbon chain of 1, 2 or 3 carbon atoms, and wherein the antigensare reversibly bound to antibodies specific for the antigens.
 12. Thecoated metal surface on a solid support according to claim 11, whereinthe antigens are the same or different and are bound to the same proteinlayer or to different patches of protein layers and are selected fromthe group consisting of optionally derivatized explosives and narcotics.13. The coated metal surface on a solid support according to claim 12,wherein the explosives are selected from the group consisting oftrinitrotoluene (TNT), dinitrotoluene (DNT),hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX),octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazine (HMX), pentaerythritoltetranitrate (PSTN), and nitroglycerine (NG).
 14. The coated metalsurface on a solid support according to claim 12, wherein the narcoticsare selected from the group consisting of cocaine, heroine, amphetamine,methamphetamine, cannabiols, tetrahydrocannabiols (THC), andmethylenedioxy-N-methylamphetamine (Ecstacy).
 15. The coated metalsurface on a solid support according to claim 11, wherein the solidsupport is a piezoelectric crystal electrode or a glass plate or prism,the antibodies are more weakly bound to the immobilized antigens than toan analyte antigen to be tested for by displacement of the antibody fromthe immobilized antigen.
 16. The coated metal surface of clam 15,wherein the antibodies are monoclonal antibodies produced with the sameimmobilized antigen linked by a longer linker than the 1-3 carbon atomlinker for the coating of the coated metal surface to Keyhole LimpetHemocyanin (KLH).
 17. The coated metal surface of claim 16, wherein theantibody has sub-nanomolar affinity to the antigen.